Small-vessel-diseases are a leading cause of Vascular Dementia. Our long-term goal is to elucidate the signaling pathways that control the structural and functional integrity of small arteries and understand the connections between these pathways and the development of small-vessel-diseases, as a prerequisite to the development of therapeutics. The research we propose is focused on the NotchS signaling pathway. Notch signaling is an evolutionary conserved pathway that plays a central role in the development and maturation of most vertebrate organs. We identified NotchS as the causative gene of CADASIL, an increasingly recognized autosomal dominant form of systemic small-vessel-disease causing stroke and dementia. We previously showed that 1[unreadable]) CADASIL patients carry highly stereotyped missense mutations leading to an odd number of cysteine residues within the extracellular domain of NotchS; 2[unreadable]) NotchS expression is largely confined to small arteries and vascular Smooth Muscle Cells (vSMC) and 3[unreadable]) Mice expressing an archetypal CADASIL mutation (R90C) targeted in vSMC develop features of the CADASIL arteriopathy. Our specific hypothesis is that appropriate level of NotchS activity is critical for structural and functional integrity of small arteries by modulating an RBP-JK dependent, Hes/HEY independent pathway. That hypothesis is supported by our recent findings: 1 [unreadable]) In adult NotchS null mice, small arteries exhibit structural defects and cerebrovascular reactivity is strongly defective. Notably, NotchS null and CADASIL phenotypes are very different; 2[unreadable]) RBP-JK dependent activity is abolished in arteries of NotchS null mice but expression level of Hes/HEY genes is unaffected. Here we propose three specific aims to further our understanding of NotchS- dependent small-vessel-diseases and NotchS signaling and address a major unresolved issue: the extent to which CADASIL mutations impair NotchS activity. We will construct and analyze NotchS gain-of-function mutant mice to determine effect of increasing NotchS activity in small arteries (Aim #1). We will determine the gene expression signature of loss and gain-of-function alleles of NotchS and identify direct target genes of NotchS by microarray analysis on isolated small arteries (Aim # 2). We will investigate effect of the archetypal R90C CADASIL mutation on NotchS wildtype activity in vivo using our Notch3R90C mice, NotchS null mice as a "rescue" system and transgenic RBP-JK reporter mice (Aim # 3).